Development of strategies towards the cryopreservation of germplasm of Ekebergia capensis Sparrm. : an indigenous species that produces recalcitrant seeds.

View/Open

Date

Author

Metadata

Abstract

The conservation of germplasm of indigenous plant species is vital not only to preserve
valuable genotypes, but also the diversity represented by the gene pool. A complicating
factor, however, is that a considerable number of species of tropical and sub-tropical
origin produce recalcitrant or otherwise non-orthodox seeds. Such seeds are hydrated
and metabolically active when shed and cannot be stored under conventional conditions
of low temperature and low relative humidity. This poses major problems for the longterm
conservation of the genetic resources of such species. Presently, the only strategy
available for the long-term conservation of species that produce recalcitrant seeds is
cryopreservation.
Ekebergia capensis is one such indigenous species that produces recalcitrant seeds. The
aim of the present study was to develop methods for the cryopreservation of germplasm
of this species. Different explant types were investigated for this purpose, viz.
embryonic axes (with attached cotyledonary segments) excised from seeds, and two in
vitro-derived explants, i.e. ‘broken’ buds excised from in vitro-germinated seedlings
and adventitious shoots generated from intact in vitro-germinated roots. Suitable
micropropagation protocols were developed for all explant types prior to any other
experimentation.
Before explants could be cryopreserved it was necessary to reduce their water content in
order to limit damaging ice crystallisation upon cooling. All explants tolerated
dehydration (by flash drying) to 0.46 – 0.39 g gˉ¹ water content (dry mass basis) with
survival ranging from 100 – 80%, depending on the explant. In addition, penetrating
and non-penetrating cryoprotectants were used to improve cryo-tolerance of explants.
The cryoprotectants tested were sucrose, glycerol, DMSO and a combination of sucrose
and glycerol. Explant survival following cryoprotection and dehydration ranged from
100 – 20%. Cryoprotected and dehydrated explants were exposed to cryogenic
temperatures by cooling at different rates, since this factor is also known to affect the
success of a cryopreservation protocol. The results showed that ‘broken’ buds could not
tolerate cryogen exposure. This was likely to have been a consequence of the large size
of explants and their originally highly hydrated condition. Adventitious shoots tolerated
cryogenic exposure slightly better with 7 – 20% survival after cooling in sub-cooled
nitrogen. Limited shoot production (up to 10%) was obtained when axes with attached
cotyledonary segments were exposed to cryogenic temperatures. In contrast, root
production from axes cooled in sub-cooled nitrogen remained high (67 – 87%).
Adventitious shoots were subsequently induced on roots generated from cryopreserved
axes by applying a protocol developed to generate adventitious shoots on in vitrogerminated
roots. In this manner, the goal of seedling establishment from cryopreserved
axes was attained.
Each stage of a cryopreservation protocol imposes stresses that may limit success. To
gain a better understanding of these processes the basis of damage was investigated by
assessing the extracellular production of the reactive oxygen species (superoxide) at
each stage of the protocol, as current thinking is that this is a primary stress or injury
response. The results suggested that superoxide could not be identified as the ROS
responsible for lack of onwards development during the cryopreparative stages or
following cryogen exposure.
The stresses imposed by the various stages of a cryopreservation protocol may affect the
integrity of germplasm. Since the aim of a conservation programme is to maintain
genetic (and epigenetic) integrity of stored germplasm, it is essential to ascertain
whether this has been achieved. Thus, explants (axes with cotyledonary segments and
adventitious shoots) were subjected to each stage of the cryopreservation protocol and
the epigenetic integrity was assessed by coupled restriction enzyme digestion and
random amplification of DNA. The results revealed little, if any, DNA methylation
changes in response to the cryopreparative stages or following cryogen exposure.
Overall, the results of this study provided a better understanding of the responses of
germplasm of E. capensis to the stresses of a cryopreservation protocol and two explant
types were successfully cryopreserved. Future work can be directed towards elucidating
the basis of damage incurred so that more effective protocols can be developed.
Assessment of the integrity of DNA will give an indication as to the suitability of
developed protocols, or where changes should be made to preserve the genetic (and
epigenetic) integrity of germplasm.